In order for estrogen receptor (ER) to activate gene transcription it must overcome a repressive chromatin structure. ER does this by recruiting to the promoter enzymes that either covalently modify chromatin or remodel it into a more permissive state. Only recently has it become apparent that all chromatin is not homogenous. Histone variants replace their canonical histone counterparts in specific regions of the genome. The effect of histone variants on gene regulation is largely unknown. However, preliminary data indicates that the histone variant macroH2A is involved in ER-regulated transcription in breast cancer cells. MacroH2A has also been implicated in the epigenetic silencing of transcription that occurs during X inactivation. Recently, macroH2A was shown to bind to the nicotinamide adenine dinucleotide (NAD+) metabolites ADP-ribose (ADPR) and O-acetyl-ADP-ribose (OAADPR). This finding implicates macroH2A as a target of exciting signaling events in two NAD+-dependent pathways: the turnover of poly(ADP-ribose) polymerase (PARP)-synthesized poly(ADP-ribose) (PAR) chains by poly(ADP-ribose) glycohydrolase (PARC) and deacetylation of proteins by SIRT1. The objective of these studies is to achieve a better understanding of both the causes of macroH2A incorporation at estrogen target genes (Aim 1) and effects of macroH2A incorporation at these genes (Aim 2). Toward this end, a variety of biochemical cell-based,and bioinformatic approaches will be used including Chromatin immunoprecipition (ChIP), ChlP-chip, expression analysis, RNAi, protein-protein interaction assays, enzyme assays, and DMA motif finding algorithms. Estrogen signaling through the ER plays important roles in normal physiological processes such as sexual development and fertility. However, estrogen also promotes the growth and progression of breast cancer and has a protective effect from heart disease, stroke and osteoporosis. Therefore, an understanding of the mechanism by which macroH2A regulates the transcription of ER target genes will provide insights into the progression of these diseases. [unreadable] [unreadable] [unreadable]